Fixable viability dye efluor 780

Manufactured by BioLegend

Sourced in United States

Fixable Viability Dye eFluor 780 is a fluorescent dye that can be used to distinguish between live and dead cells in flow cytometry applications. It binds to proteins with amine groups, allowing it to permanently stain dead cells. The dye has an excitation maximum at 780 nm and an emission maximum at 810 nm, making it compatible with the common 785/805 nm laser and filter set.

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Lab products found in correlation

Monensin, by Thermo Fisher Scientific (2 mentions) Lsrfortessa flow cytometer, by BD (2 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Thermo Fisher Scientific (1 mentions) Anti cd107a, by BioLegend (1 mentions) Lag 3 pe, by BioLegend (1 mentions) Cd8 pe clone rpa t8, by BioLegend (1 mentions) Lsrfortessa, by BD (1 mentions) Cd8 pe cy7, by BD (1 mentions) Cd3 pacific blue clone ucht1, by BD (1 mentions) Apoptosis detection kit, by BioLegend (1 mentions) Pd 1 29f 1a12, by BioLegend (1 mentions) Thy1.1 ox 7, by BioLegend (1 mentions) T bet ebio4b10, by BioLegend (1 mentions)

Close spelling variants of this product

Fixable viability dye (27 citations) Viability dye (15 citations) EFluor 780 (8 citations)

7 protocols using fixable viability dye efluor 780

Multiparametric Analysis of Activated B Cells

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PBMC cultures were performed as described. A cell stimulation cocktail (eBioscience, Thermo Fisher Scientific) was added to the culture for the last 4 h of culturing. After 24 h, cells were stained with Fixable Viability Dye eFluor™ 780 and the following antibodies purchased from BioLegend or BD BioScience: CD3 (HIT3a, PerCP-Cy5.5), CD20 (2H7, BV510), CD24 (ML5, BV650), CD27 (O323, BV421), CD38 (HIT2, PE/Dazzle), and IgD (IA6-2, FITC). Fixation and permeabilization were performed using an IC Fix and Perm Buffer Kit (eBioscience, Thermo Fisher Scientific). Anti-human IL-6 (MQ2-13A5, PE-Cy7) was used to detect intracellular cytokine levels in B cells.

Einhaus J., Pecher A.C., Asteriti E., Schmid H., Secker K.A., Duerr-Stoerzer S., Keppeler H., Klein R., Schneidawind C., Henes J, & Schneidawind D. (2020). Inhibition of effector B cells by ibrutinib in systemic sclerosis. Arthritis Research & Therapy, 22, 66.

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Intracellular Cytokine Staining and ELISA Antibody Quantification

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For intracellular cytokine staining, cryopreserved PBMCs were stimulated with 10 μg/ml PPD (Statens Serum Institut, Copenhagen, Denmark) or phorbol myristate acetate and ionomycin (eBioscience) for 6 hours in the presence of anti-CD107a (BioLegend), brefeldin A and monensin (eBioscience). Cells were stained with various combinations of fluorochrome-labeled antibodies and Fixable Viability Dye eFluor 780 (purchased from BioLegend or eBioscience). All samples were acquired by LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
Antibody abundance was quantitated by the ELISA. 384-well high binding plates (Corning) were coated with Mtb protein antigens (31 (link)) at 2 μg/ml, or PPD, BCG lysate, and Mtb lysate at 6 μg/ml. Serum samples (1:500 dilution) were incubated overnight at 4°C. Plates were washed, followed by the addition of recombinant Protein-G HRP and incubated for 1 hour at room temperature. Plates were washed and developed with SureBlue TMB Peroxidase Substrate (KPL Inc.) followed by 1N H₂SO₄. Optical densities (OD) were read at 450 nm and 570 nm. Antibody levels were calculated by (OD450 nm - OD570 nm) subtracted from control wells of antigen and HRP alone.

Barber D.L., Sakai S., Kudchadkar R.R., Fling S.P., Day T.A., Vergara J.A., Ashkin D., Cheng J.H., Lundgren L., Raabe V.N., Kraft C.S., Nieva J.J., Cheever M.A., Nghiem P.T, & Sharon E. (2019). Tuberculosis following PD-1 blockade for cancer immunotherapy. Science translational medicine, 11(475), eaat2702.

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Multiparameter Phenotyping of Transduced T Cells

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Transduced CD8⁺ T cells were stained for 15 min at 4°C with fluorescently labeled antibodies against CD3-Pacific Blue (clone UCHT1, ref. 558117; BD) and CD8-PE (clone RPA-T8, ref. 301008; BioLegend); Fixable Viability Dye eFluor 780 (ref. 65-0865-14; Thermo Fisher Scientific) was used to evaluate transduction efficiency. Fresh, expanded, and transduced cells were stained with Fixable Viability Dye eFluor 780 (ref. 65-0865-14; Thermo Fisher Scientific), CD62L-BV650 (clone DREG-56, ref. 2124160; Sony Biotechnology), CD25-PerCP Cy5.5 (clone BC96, ref. 2113130; Sony Biotechnology), TIM3-APC (clone 34482, ref. FAB2365A; R&D Systems [R&D]), CD3-AF700 (clone UCHT1, ref. 300424; Sony Biotechnology), CD45RA-BV421 (clone HI100, ref. 304118; BioLegend), CD45RO-BV711 (clone UCHL1, ref. 304236; BioLegend), LAG3-PE (ref. FAB2319P; R&D), CD8-PE-Cy7 (clone SK1, ref. 335822; BD), TCR Vβ21.3 (clone IG125, ref. PN IM1483; Beckman Coulter), and CD19 CAR detection reagent (ref. AB_2811310; Miltenyi Biotec). Flow cytometry analysis was performed on a BD LSRFortessa using FACSDiva, with acquisition of a fixed number of cells for each experiment. FACS data analysis was performed using FlowJo version (v.)10 . Data are presented as percent of cells and as MFI.

Lo Presti V., Cornel A.M., Plantinga M., Dünnebach E., Kuball J., Boelens J.J., Nierkens S, & van Til N.P. (2021). Efficient lentiviral transduction method to gene modify cord blood CD8+ T cells for cancer therapy applications. Molecular Therapy. Methods & Clinical Development, 21, 357-368.

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Neuropilin-1 Expression and Dp1 Binding Assay

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DC2.4 cells (4 × 10⁴ cells) were cultured in 96-well plates for 12 h at 37°C. The cells were further incubated with 10 μM NRP-1 siRNA (5’-ctgctacttcacagtatggta-3’) and 0.4 μL Lipofectamine 3000 reagent (ThermoFisher) for 48 h at 37°C. Subsequently, the cells were collected and analyzed for NRP-1 expression and Dp1 binding using flow cytometry. NRP1 expression was evaluated using Fixable Viability Dye eFluor™ 780 and BV421-labelled anti-mouse CD304 (Neuropilin-1) antibody (BioLegend).

Matsuda T., Misato K., Tamiya S., Akeda Y., Nakase I., Kuroda E., Takahama S., Nonaka M., Yamamoto T., Fukuda M.N, & Yoshioka Y. (2022). Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice. iScience, 25(11), 105324.

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Multiparameter Flow Cytometric Analysis of T Cell Checkpoint Receptors

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Flow cytometric analyses were used to determine the cell surface expression of ICs including, PD-1, TIM-3 and LAG-3, on T cell subsets in the absence of mAb treatment or following the single and combined blockade of PD-L1 and PD-1.
Cells were washed in phosphate-buffered saline (PBS), and re-suspended in 100 µL of staining buffer (PBS with 2% FCS and 0.1% sodium azide). Cells were blocked with a human IgG1 antibody (Sigma-Aldrich) for 10 min on ice. To gate out dead cells, Fixable Viability Dye eFluor 780 (FVD780; BioLegend, California, USA) was utilized. For surface staining, cells were stained with anti-CD4-Alexa Fluro 700 (Clone RPA-T4, BD Pharmingen, California, USA), anti-CD25-Brilliant Violet 650 (Clone M-A251, BioLegend), anti-PD-1-Phycoerythrin/Texas Red (PE-Dazzle^™ 594) (Clone EH12.2H7, BioLegend), anti-TIM-3-Brilliant Violet 711 (Clone 7D3; BD Biosciences, California, USA), and anti-LAG-3-Brilliant Violet 421 (Clone T47-530; BD Biosciences) for 30 min at 4 °C in the dark.

Saleh R., Toor S.M., Khalaf S, & Elkord E. (2019). Breast Cancer Cells and PD-1/PD-L1 Blockade Upregulate the Expression of PD-1, CTLA-4, TIM-3 and LAG-3 Immune Checkpoints in CD4+ T Cells. Vaccines, 7(4), 149.

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Quantifying Antigen-Specific CD8+ T Cell Killing

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Antigen-specific CD8⁺ cells were stimulated for 7 days by peptide-pulsed moDCs mentioned above. Target T2 cells (expressing HLA-A2) were divided into 2 populations, labeled with 2 different intensities of CTV. CTV_hi cells were pulsed with cognate peptides, while the CTV_lo portion remained unpulsed. CTV_hi and CTV_lo cells were mixed at a 1:1 ratio before adding to effector cells. After harvesting the antigen-specific T cells, target T2 cells containing the 2 groups were added at various effector/target ratios and incubated for 8 hours. Cell density was adjusted to 10,000 cells/well in a 96-well plate. The results were obtained by flow cytometry, and the specific killing ability was calculated by the ratio of CTV_hi to CTV_lo cells. Fixable Viability Dye eFluor 780 was used for viability assay, and Apoptosis Detection Kit (BioLegend) was used to measure cisplatin-induced apoptosis.

Sung B.Y., Lin Y.H., Kong Q., Shah P.D., Glick Bieler J., Palmer S., Weinhold K.J., Chang H.R., Huang H., Avery R.K., Schneck J, & Chiu Y.L. (2022). Wnt activation promotes memory T cell polyfunctionality via epigenetic regulator PRMT1. The Journal of Clinical Investigation, 132(2), e140508.

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ESAT-6 Peptide Stimulation of T Cells

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For T cell stimulations, cells were incubated with 5 μg/ml ESAT-6_1–20 peptide for 5 h at 37°C in the presence of brefeldin A and 1 mM aminoguanidine. Cells were stained with various combinations of the following fluorochrome-labeled antibodies: anti-CD4 (RM4-4), CD44 (IM7), CD45 (IM7), CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), CXCR3 (CXCR3-173), CX3CR1 (polyclonal), Foxp3 (FJK-16s), ICOS (15F9), IFN-γ (XMG1.2), KLRG1 (2F1/KLRG1), PD-1 (29F.1A12), T-bet (eBio4B10), Thy1.1 (OX-7), and Fixable Viability Dye eFluor 780 purchased from BioLegend, eBioscience (San Diego, CA), BD Biosciences (San Jose, CA), and R&D Systems (Minneapolis, MN). I-A^b ESAT-6_4–17 and I-A^b EsxG_46-61 MHC tetramers were produced by the NIAID Tetramer Core Facility (Emory University, Atlanta, GA). For staining with MHC class II tetramers, cells were incubated with tetramer at 1:50 dilution in complete medium containing 10% FCS, 1 mM aminoguanidine and monensin (eBioscience) at a 1:1000 dilution for 1 h at 37°C prior to staining with surface antibodies. All samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Sakai S., Kauffman K.D., Sallin M.A., Sharpe A.H., Young H.A., Ganusov V.V, & Barber D.L. (2016). CD4 T Cell-Derived IFN-γ Plays a Minimal Role in Control of Pulmonary Mycobacterium tuberculosis Infection and Must Be Actively Repressed by PD-1 to Prevent Lethal Disease. PLoS Pathogens, 12(5), e1005667.

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